CN110218670A - One plant of selenium oxidized form rhizobium T3F4 and its application with good colonization ability - Google Patents
One plant of selenium oxidized form rhizobium T3F4 and its application with good colonization ability Download PDFInfo
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Abstract
The invention belongs to field of agricultural microbial technology, specifically disclose one plant of selenium oxidized form rhizobium T3F4 and its application with good colonization ability, and the deposit number of the bacterial strain is CCTCC NO:M 2019057.The bacterial strain has oxidation to elemental selenium, fluorescent marker is carried out to the bacterial strain, is inoculated in potting soil, at 21 days, cell quantity of the bacterial strain in rhizosphere soil and pakchoi leaf is maintained at 6,600,000/g(dry weight) and 200,000/g(fresh weight), show very strong colonization ability.On the other hand, elemental selenium is added in potting or the content of effective selenium in soil can be improved in sodium selenite, T3F4, and significantly improve the Se content at each position of pakchoi.Therefore, rhizobium T3F4 can be prepared into plant selenium-rich agent, have a good application prospect in terms of biology promotes plant selenium-rich.
Description
Technical field
The invention belongs to field of agricultural microbial technology, are related to one plant of selenium oxidized form rhizobium with good colonization ability
T3F4 and its application, the rhizobium are classified as Rhizobium sp., which has outstanding fixed in soil and plant
It grows ability, increase soil available Se content, and promote plant selenium-rich.
Background technique
Selenium (Se) is located at the VIth main group of the periodic table of elements, and selenium is in nature with Se (- II), Se (0), Se (IV) and Se
(VI) form of four kinds of valence states exists.Se (- II) is mainly organic selenium, is common in organism, such as selenium important in organism
For cysteine (SeCys), selenomethionine (SeMet), monomethyl selenium (CH3- Se), dimethyl-selenide (CH3-Se-CH3) and first
Base selenocysteine (SeMeCys) etc..Se (IV) and Se (VI) is mainly inorganic selenium, is present in inorganic matter more, usually with
Oxo-anions exist, such as selenite (SeO3 2-) and selenate (SeO4 2-), it is the main source that plant absorption utilizes selenium.Change
Elemental selenium Se (0) is more rare in nature, and majority is present in selenium-rich mine.Wherein change of the selenite in these four valence states
It is most strong to close object Poisoning, and elemental selenium is substantially nontoxic.
Selenium is microelement necessary to human body, most important to human health.Human body has 25 kinds of selenoproteins, and selenium is its work
Property center, human body energy metabolism and gene expression in play an important role, such as anti-oxidant, immunological regulation and antagonism.Selenium exists
The intracorporal secure threshold of people is very narrow, and daily selenium intake will selenium deficiency lower than 40 μ g；It may be poisoned greater than 400 μ g.It lacks
Selenium will lead to more than the 40 kinds of diseases such as cardiomyopathy, immunity degradation, cognitive ability decline and Kaschin-Beck disease；But selenium excess intake also can
Lead to alopecia, loss of finger-nail, increase diabetes risk etc..
Area reaches 72% to Chinese soil selenium deficiency (being lower than 0.6mg/kg), to cause 2/3 population selenium insufficiency of intake.People
Body selemium nutrition is mainly derived from food plant, and promoting selenium intake level by diet is considered as current safest selenium-supply
Mode, thus the research for improving crop Se content is concerned.
At present there are many ways to fortification of plants selenium-rich, be most widely used be by soil spraying selenium, Foliar application of selenium and
The modes such as seed dressing.Containing elemental selenium, selenite, selenate and organic selenium etc. in soil, plant mainly absorb selenite and
Selenate, but selenite is easy to be retained by adsorption by soil, to be difficult to be absorbed and used by plants.Therefore, it adds in the soil
Microorganism makes it dissolve selenium or selenium oxide, and then increases the absorption of Selenium By The Plants, is a kind of safe and reliable, low-cost side
Formula.
Summary of the invention
The purpose of the present invention is to provide the one plant selenium oxidized form rhizobium T3F4 with good colonization ability, the bacterial strain by
Applicant separates from the selenium-rich soil of enshi, and the Strain Designation is Rhizobiunsp.T3F4, and deposit number is
CCTCC NO:M 2019057.
It is another object of the present invention to provide application of the rhizobium T3F4 in selenolite.
Final object of the present invention is the provision of application of the rhizobium T3F4 in preparation plant selenium-rich agent, soil
Middle addition microbial inoculum T3F4 can promote pakchoi to absorb selenium, and Se content when mature in pakchoi significantly rises, wherein in blade
Se content rises up to 43.1%, and root Se content rises up to 60.2%.In addition, rhizobium T3F4 can also promote soil Se form
Conversion, the effective Se content of potting soil rises up to 9.9% when pakchoi maturation.
In order to achieve the above object, the present invention takes following technical measures:
Applicant separates from Chinese Enshi State of Hubei Province selenium-rich soil, screens one plant have oxidizing simple substance selenium ability and
Promote the rhizobium T3F4 of pakchoi selenium-rich in the soil, which is named as rhizobium (Rhizobium sp.) by applicant
T3F4, the bacterial strain have been sent on January 18th, 2019 to China typical culture collection center (CCTCC) preservation, deposit number
For CCTCC NO:M 2019057, classification naming: Rhizobium sp.T3F4.
The specific features of the bacterial strain are as follows:
A. morphological feature
The cellular morphology of rhizobium T3F4 be it is rod-shaped, it is 1-1.25 μm long, it is 0.2-0.25 μm wide.
B. cultural characteristic
Rhizobium T3F4 28 DEG C of culture 48h on 1/5LB Agar Plating, form the faint yellow petite of round shape,
The smooth of the edge, protuberance.
The difficult elemental selenium using state is oxidized to water-soluble by application of the rhizobium T3F4 in selenolite including the use of the bacterial strain
The selenite of state.
Application of the rhizobium T3F4 in preparation plant selenium-rich agent, which is sprinkled into soil, can not only be promoted
The elemental selenium of not solvent is oxidized to the selenite that plant available utilizes, can also convert the selenium in soil by plant selenium-rich
Form allows plant more effectively to utilize selenium element.
Compared with prior art, the invention has the following advantages that
(1) one plant of rhizobium T3F4 that the present invention obtains has the elemental selenium of not solvent being oxidized to plant available benefit
The ability of selenite, to promote plant selenium-rich；It is the supplement to extremely limited selenolite strain resource.
(2) rhizobium T3F4 of the invention stable in the soil can be colonized, and have the ability of solution selenium, and the selenium of indissoluble state is turned
The selenium using state is turned to, plant absorption selenium is promoted.
(3) rhizobium T3F4 of the invention is a kind of endophyte of plant, can be colonized for a long time in plant roots, in blade,
Sustainability is strong, the long-term effect for playing selenium oxide reconciliation selenium.
(4) safe and environment-friendly the present invention provides a kind of new paragon for promoting plant selenium-rich using beneficial microbe.
Detailed description of the invention
Fig. 1 is the schematic diagram that elemental selenium is oxidized to selenite by rhizobium T3F4；
The processing that Se (0) expression only adds elemental selenium that rhizobium T3F4 is not added in Fig. 1 compares, and Se (0)+T3F4 indicates both to add root
Tumor bacterium T3F4 adds the experiment process of elemental selenium again.
Fig. 2 is the effect picture that pakchoi absorbs selenium after adding rhizobium T3F4 processing；
Upper figure is potting pakchoi root Se content in Fig. 2, and Fig. 2 following figure is potting pakchoi blade Se content；Control
Selenium processing is not added in expression；+ T3F4 is indicated plus rhizobium T3F4 processing；- T3F4 indicates the blank control that bacterium is not added；Se (0) is indicated
Elemental selenium is added to handle；Se (IV) is indicated plus sodium selenite processing.
Fig. 3 is influence of the rhizobium T3F4 to pakchoi potting soil Se form；
Selenium processing is not added in Control expression；+ T3F4 is indicated plus rhizobium T3F4 processing；- T3F4 indicates the blank that bacterium is not added
Control；Se (0) is indicated plus elemental selenium processing；Se (IV) is indicated plus sodium selenite processing；0d indicates the primitive soil for not planting pakchoi
Earth；21d indicates the soil after plantation pakchoi 21 days.
Fig. 4 is that rhizobium T3F4 colonizes quantity in rhizosphere soil, non-rhizosphere soils, plant leaf blade；
T3F4 Rhizosphere Soil indicates the rhizosphere soil for adding rhizobium T3F4 in Fig. 4；T3F4+Se (IV) Rhizosphere Soil indicates plus root nodule
Bacterium T3F4 adds the rhizosphere soil of sodium selenite；T3F4+Se (0) Rhizosphere Soil indicates the Rhizosphere Soil for adding rhizobium T3F4 to add elemental selenium
Earth；T3F4 Non-rhizosphere soil indicates plus the non-rhizosphere soils of rhizobium T3F4；T3F4+Se (IV) Non-rhizosphere soil indicates plus rhizobium
T3F4 adds the non-rhizosphere soils of sodium selenite；T3F4+Se (0) Non-rhizosphere soil indicates the non-rhizosphere for adding rhizobium T3F4 to add elemental selenium
Soil；T3F4 blade indicates plus the pakchoi blade of rhizobium T3F4；T3F4+Se (IV) blade indicates plus rhizobium T3F4 adds Asia
The pakchoi blade of sodium selenate；T3F4+Se (0) blade indicates the pakchoi blade for adding rhizobium T3F4 to add elemental selenium.
Specific embodiment
The present invention is described in further details combined with specific embodiments below.Embodiment is being with technical solution of the present invention
Under the premise of implemented, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to
Following embodiments.Method therefor is conventional method unless otherwise instructed in following embodiments.
Embodiment 1: separation, the identification of rhizobium (Rhizobium sp.) T3F4
(1) sample is taken: applicant is in July, 2012 from Hubei Province Enshitujianationalitymiaonationality Autonomous Prefecture selenium-enriched area surface layer
Selenium-rich soil sample used in the present invention is collected in soil.
(2) thallus isolates and purifies: weighing soil sample 10g in the triangular flask equipped with 90ml sterile saline, sets 28 DEG C and shake
Half an hour is vibrated in bed, then successively takes 1ml to be added in 9ml sterile saline and is gradually diluted to 10-2, 10-3, 10-4, 10-5,
0.1ml is taken to be coated in 1/5LB solid medium respectively, each gradient applies 3 plates.Set 28 DEG C of constant temperature incubations one week, picking
Single colonie scribing line purifying.After purification by 50% glycerol: bacterium solution (volume ratio 1:1) is stored in screw socket pipe, -80 DEG C of preservations.
(3) screening of selenium oxidation bacteria: 0.1g simple substance selenium powder (CP) Yu Hanyou 100ml 1/5LB fluid nutrient medium is weighed
In the triangular flask of 250ml, 115 DEG C of sterilizing 20min.After cooling, access bacterium solution 150 μ l, 28 DEG C, 160rpm shaking table culture 6d, in
Between spot sampling (supernatant).Gained supernatant is centrifuged 10min in 8000rpm, uses atomic fluorescence morphological analysis instrument detection four
The content of valence selenium.If tetravalence Se content obviously increases, illustrate that the bacterium has oxidisability to elemental selenium.
(4) taxonomic identification of selenium oxidation bacteria: carrying out 16S rRNA gene sequencing to the bacterial strain of acquisition and construct its system into
Change tree, in conjunction with its physiological and biochemical property, applicant identifies the Pseudomonas in rhizobium.The bacterial strain was sent on 01 18th, 2019
Toward China typical culture collection center (CCTCC) preservation, classification naming: Rhizobiunsp.T3F4, deposit number CCTCC
NO:M 2019057, address: Wuhan, China Wuhan University.
The specific features of the bacterial strain are as follows:
A. morphological feature
The cellular morphology of rhizobium T3F4 be it is rod-shaped, it is 1-1.25 μm long, it is 0.2-0.25 μm wide.
B. cultural characteristic
Rhizobium T3F4 28 DEG C of culture 48h on 1/5LB Agar Plating, form the faint yellow petite of round shape,
The smooth of the edge, protuberance.
Embodiment 2: rhizobium T3F4 oxidizing simple substance selenium curve
It is described that specific step is as follows:
(1) prepare 3 bottles of sterilized 1/5LB fluid nutrient mediums, every bottle of 100ml.Three bottles of each 0.5g/L that are added are taken to sterilize
Elemental selenium.It is inoculated with logarithmic phase (OD600Value about 0.8) T3F4 bacterium solution, inoculum concentration 1%.2ml is sampled after mixing at once, the sample conduct
It samples, is placed in clean centrifuge tube for the first time.Each triangular flask is placed in 28 DEG C, is cultivated in 160rpm constant-temperature table.Sample time
Respectively 0d, 1d, 2d, 4d, 5d, 6d, the experimental setup three repeat.Sample makees following processing after sampling.
(2) the sample 2ml, 12000rpm taken every time is centrifuged 10min, and supernatant crosses 0.22 μm of filter membrane in -20 DEG C of refrigerators
It saves.
(3) with Se in Beijing Jitian Instrument Co., Ltd.'s atomic fluorescence morphological analysis instrument (model SA-20) test sample
(IV) content.
(4) to measure Se (IV) concentration (μm ol/L) as ordinate, the time is that abscissa constructs elemental selenium oxidation curve graph
(Fig. 1).
(5) as shown in Figure 1, Se (IV) content is in fall before rear ever-increasing trend in sample supernatant.0th day to
1 day, Se (IV) content in supernatant declined rapidly, dropped to 1.1 μm of ol/L from 2.5 μm of ol/L.From the 1st day to the 6th day, Se
(IV) content is continuously increased, and until the 6th day, Se (IV) content is 3.26 μm of ol/L in supernatant.
Embodiment 3:
Rhizobium T3F4 promotes pakchoi selenium-rich and Selenium in Soil Forms Transformation
It is described that specific step is as follows:
1 experimental group of table
"+" indicates that addition, "-" expression are not added
Pakchoi: Chinese holly pakchoi is purchased from Jiangxi Province Fengcheng City Hang Chengzhong industry Co., Ltd
(1) by wind desiceted soil weighing 2kg/ basin, sodium selenite or elemental selenium (5mg/kg, chemical combination then soil pretreatment: are applied
Object concentration), it mixes thoroughly, water is added by 15% moisture content, balances 3 days；
(2) after the completion of balancing, the rhizobium T3F4 mother liquor saved with physiological saline is diluted with water, equably by rhizobium
T3F4 bacterium solution is sprayed at native surface, guarantees bacterial content > 10 in soil7Cfu/g, soil are mixed thoroughly, 12 repetitions of each processing；
(3) the consistent Chinese holly pakchoi of growing way (plant height about 6cm), 1 plant of pakchoi of every basin are transplanted after soil is mixed thoroughly immediately
Seedling；
(4) sufficient moisture is kept by Chinese cabbage growth demand after transplanting；
(5) pakchoi is mature after transplanting 21 days, samples (plant complete stool) and soil；
(6) after sample water-removing is according to common tea-manufacturing technology, drying (to 5% or less moisture content), crushing, by the Chinese people
Republic standard GB/T 500993-2017 " method for measuring of selenium in food " clears up Plant samples；The survey of soil Se form
It is fixed that pedotheque (second lieutenant Wu etc., 2004) is extracted according to " the soil Se form extracting method " of the reports such as second lieutenant Wu.
(7) it is measured and is planted with the atomic fluorescence morphological analysis instrument (model HG-AFS) that Beijing Jitian Instrument Co., Ltd. produces
Selenium in strain and soil.
(8) result data tested SPSS Statistics22.0 statistical software is handled, with plant or soil list
The Se content (mg/kg) of position weight is ordinate, and each processing is abscissa, and 8 software of Origin does figure, as a result such as Fig. 2 institute
Show.
(9) as shown in Fig. 2, soil addition elemental selenium or sodium selenite can significantly improve pakchoi, each position selenium contains
Amount, the processing group for adding T3F4 are compared with corresponding control group, and each position Se content of pakchoi has different degrees of rising.
T3F4 processing group is compared with control group (Control), root Se content rising 37.3%, respectively 0.75mg/kg and 1.03mg/
kg；Blade Se content rising 47.2%, respectively 0.570mg/kg and 0.839mg/kg.Se (0)+T3F4 processing group and Se (0)
Control group is compared, and root Se content rises 26.3%, respectively reaches 3.38mg/kg and 4.27mg/kg；Blade Se content rises
31.1%, respectively reach 1.739mg/kg and 2.279mg/kg.Se (IV)+T3F4 processing group is compared with Se (IV) control group,
Root Se content rises 60.2%, respectively reaches 2.45mg/kg and 3.92mg/kg；Blade Se content rises 43.1%, reaches respectively
To 1.499mg/kg and 2.145mg/kg.
(10) as shown in figure 3, addition microbial inoculum T3F4 can significantly improve the accounting of effective selenium in soil, after planting pakchoi
Soil compare effective selenium (water-soluble state and exchangeable species) accounting with initial soil and have different degrees of rising.Pakchoi is mature
When, compared with the control group, effective Se content rises to 46.2% from 36.3% to T3F4 processing group；T3F4+Se (IV) processing group and
Se (IV) control group is compared, and effective Se content rises to 38.9% from 29.0%；T3F4+Se (0) processing group and Se (0) control group
It compares, effective Se content rises to 20.4% from 15.7%.
Embodiment 4:
Colonization dynamics of the T3F4 in Rhizosphere Soil, Non-rhizosphere soil and blade
(1) plasmid pHC60 (containing GFP) fluorescent marker rhizobium T3F4: is transformed by root using this hybridizing method of parents
In tumor bacterium T3F4, the expression of green fluorescence is verified under fluorescence microscope, the rhizobium T3F4 for being successfully transferred to GFP is named as
T3F4-GFP。
(2) by wind desiceted soil weighing 2kg/ basin, sodium selenite or elemental selenium (5mg/kg, chemical combination then soil pretreatment: are applied
Object concentration), it mixes thoroughly, water is added by 15% moisture content, balances 3 days.
(3) after the completion of balancing, the rhizobium T3F4 mother liquor saved with physiological saline is diluted with water, by bacterium amount > 107cfu/
Rhizobium T3F4 bacterium solution is equably sprayed at native surface by g, and soil is mixed thoroughly, 12 repetitions of each processing.
(4) the consistent Chinese holly pakchoi of growing way (plant height about 6cm), 1 plant of pakchoi of every basin are transplanted after soil is mixed thoroughly immediately.
(5) sufficient moisture is kept by Growth of Cabbage demand after transplanting.
(6) it was sampled respectively at 3 days, 7 days, 15 days and 21 days after filling bacterium, takes one plant of complete pakchoi for every group every time, surveyed
Determine colonization dynamics of the T3F4-GFP in Rhizosphere Soil and Non-rhizosphere soil.
(7) soil sample count plate.Every sub-sampling uproops the whole strain of pakchoi, shakes off and collects the soil being attached on root
Earth collects around root other than 5cm soil as Non-rhizosphere soil as Rhizosphere Soil.Take two parts of soil fresh weight 2g, a soil 65
DEG C drying to constant weight, and sterile water 18mL is added in another soil, vibrates 30min, dilutes after mixing well by 10 times of dilution methods, point
Do not take 10-3、10-4With 10-5The 100 μ L of suspension of concentration applies plate, and 3 repetitions of each concentration are coated on containing 50 Fourth Rings μ g/mL
The 1/5LB resistant panel of plain (Tetracycline) after 28 DEG C of constant temperature incubation 48h, counts resistance under inverted fluorescence microscope
Clump count on plate calculates the bacteria containing amount (cfu/g) of every gram of soil (dry weight).
(8) blade count plate.Pakchoi root and stem are cut off after every sub-sampling, after being cleaned with tap water, naturally dry table
Face moisture weighs blade 1.0g, and 0.85% NaCl 9ml is added, is fully ground, and after diluting by 10 times of dilution methods, takes respectively
10-2、10-3With 10-4The 100 μ l coated plate of suspension of concentration, 3 repetitions of each concentration are coated on containing 50 μ g/mL tetracyclines
(Tetracycline) after 28 DEG C of constant temperature incubation 48h, it is flat to count resistance under inverted fluorescence microscope for 1/5LB resistant panel
Clump count on plate counts and calculates the bacteria containing amount (cfu/g) of every gram of blade (fresh weight).
(9) calculated result data SPSS Statistics22.0 statistical software, institute is handled, with colony counts
[log (cfu/g)] is ordinate, and sample time is abscissa, and 8 software of Origin does figure, as a result as shown in Figure 4.
(10) as shown in figure 4, added in soil elemental selenium or sodium selenite to T3F4 colonizing in soil and pakchoi
Do not influence.When meeting bacterium 3d, the quantity of the T3F4-GFP in Rhizosphere Soil reaches 3 × 108Cfu/g, hereafter, the quantity of T3F4 have
Declined, has reached 2.0 × 10 in 7d7Cfu/g, 14d reach 1.2 × 107Cfu/g, in pakchoi maturation (the
21d), T3F4 concentration still can achieve 6.6 × 106Cfu/g shows that T3F4 can be colonized for a long time in pakchoi rhizosphere.In non-root
It can also be observed that similar phenomenon in border soil and pakchoi blade, in 3d, T3F4-GFP is in Non-rhizosphere soil and pakchoi
The quantity of blade is 2.1 × 106Cfu/g, and in pakchoi maturation, T3F4-GFP are in Non-rhizosphere soil and pakchoi blade
Quantity is respectively 3.2 × 105Cfu/g and 2.0 × 105cfu/g。
Claims (4)
1. the deposit number of the rhizobium of one plant of separation, the rhizobium is CCTCC NO:M 2019057, classification naming isRhizobiunsp. T3F4。
2. application of the rhizobium described in claim 1 in selenium oxide.
3. application of the rhizobium described in claim 1 in preparation plant selenium-rich agent.
4. application of the rhizobium described in claim 1 in conversion soil Se form.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322211A (en) * | 2021-07-09 | 2021-08-31 | 华中农业大学 | LX-88 bacterium with good selenium conversion and Se (0) oxidation capacity and application thereof |
| CN113322212A (en) * | 2021-07-09 | 2021-08-31 | 华中农业大学 | LX-1 strain Dyella sp and application thereof in oxidizing organic selenium or elemental selenium |
| CN113430136A (en) * | 2021-07-09 | 2021-09-24 | 华中农业大学 | Rhodanobacter sp.LX-100 strain and application thereof in oxidizing organic selenium or elemental selenium |
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| A V TUGAROVA等: "The ability of rhizobacteria Azospirillum brasilense to the selenium (IV) reduction to selenium(0)", 《MIKROBIOLOGIIA》 * |
| 成永丽等: "6 种根瘤菌***发育分析及其耐硒能力", 《西南农业学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322211A (en) * | 2021-07-09 | 2021-08-31 | 华中农业大学 | LX-88 bacterium with good selenium conversion and Se (0) oxidation capacity and application thereof |
| CN113322212A (en) * | 2021-07-09 | 2021-08-31 | 华中农业大学 | LX-1 strain Dyella sp and application thereof in oxidizing organic selenium or elemental selenium |
| CN113430136A (en) * | 2021-07-09 | 2021-09-24 | 华中农业大学 | Rhodanobacter sp.LX-100 strain and application thereof in oxidizing organic selenium or elemental selenium |
| CN113322211B (en) * | 2021-07-09 | 2022-05-20 | 华中农业大学 | LX-88 bacterium with good selenium conversion and Se (0) oxidation capacity and application thereof |
| CN113322212B (en) * | 2021-07-09 | 2022-05-20 | 华中农业大学 | LX-1 strain and application thereof in oxidizing organic selenium or elemental selenium |
| CN113430136B (en) * | 2021-07-09 | 2022-05-20 | 华中农业大学 | Rhodanobacter sp.LX-100 strain and application thereof in oxidizing organic selenium or elemental selenium |
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| CN110218670B (en) | 2020-11-24 |
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